LncRNA TUG1 relieves renal mesangial cell injury by modulating the miR‐153‐3p/Bcl‐2 axis in lupus nephritis

Abstract Background Lupus nephritis (LN) is one of the most common and serious complications of systemic lupus erythematosus. Our experiments aimed to evaluate the molecular mechanisms of long noncoding RNA (lncRNA) TUG1 in a human renal mesangial cell (HRMC) model of LN. Methods Cells were treated with lipopolysaccharide (LPS) to induce inflammatory damage. StarBase, TargetScan, and a luciferase reporter assay were used to predict and confirm the interactions between lncRNA TUG1, miR‐153‐3p, and Bcl‐2. We determined the lncRNA TUG1 and miR‐153‐3p levels in LPS‐induced HRMCs using quantitative reverse transcription PCR (RT‐qPCR). MTT and flow cytometry analyses were used to detect HRMC proliferation and apoptosis, respectively. In addition, the expression of the apoptosis‐related proteins Bax and Bcl‐2 was evaluated using western blot analysis and RT‐qPCR. Lastly, the secretion of inflammatory cytokines (IL‐1β, IL‐6, and TNF‐α) was assessed using ELISA. Results miR‐153‐3p directly targeted lncRNA TUG1. The level of lncRNA TUG1 was remarkably lower and miR‐153‐3p expression was markedly higher in LPS‐treated HRMCs than in untreated cells. Transfection with TUG1‐plasmid relieved LPS‐induced HRMC injury, as evidenced by increased cell viability, inhibited apoptotic cells, reduced Bax expression, increased Bcl‐2 level, and reduced secretion of inflammatory cytokines. Importantly, these findings were reversed by miR‐153‐3p mimic. We also found that miR‐153‐3p directly targeted Bcl‐2 and negatively regulated Bcl‐2 expression in HRMCs. In addition, our findings suggest that miR‐153‐3p inhibitor relieved LPS‐induced HRMC injury via the upregulation of Bcl‐2. Conclusion lncRNA TUG1 alleviated LPS‐induced HRMC injury through regulation of the miR‐153‐3p/Bcl‐2 axis in LN.


| INTRODUCTION
Systemic lupus erythematosus (SLE) is a typical autoimmune disease that can affect multiple systems and is caused by imbalances in the human immune system, in which the human body forms antibodies against its own organs or tissues. 1,2 The initial symptoms of SLE often involve an important organ or system, including joint swelling and pain, morning stiffness, proteinuria, and leukopenia. 3,4 Lupus nephritis (LN) is the most common complication of SLE. Approximately 60%-70% of patients with SLE develop LN. 5 The pathogenesis of LN is multifactorial, and currently there is no satisfactory strategy to prevent and treat LN.
Long noncoding RNAs (lncRNAs) are more than 200 nucleotides long with no protein-coding capacity. As vital biological regulators, lncRNAs have been reported to be involved in biological processes, including cell proliferation, apoptosis, and cell differentiation. 6,7 Studies have shown that lncRNA TUG1 regulates the proliferation and apoptosis of vascular smooth muscle cells (VSMC) and HUVEC by regulating the miR-148b/ IGF2 axis. 8 Moreover, Cao et al. 9 demonstrated the clinical significance of the reduced expression of lncRNA TUG1 in the peripheral blood of patients with SLE. Therefore, lncRNA TUG1 may serve as a clinical diagnostic marker for patients with SLE, with or without LN. However, the expression and mechanism of lncRNA TUG1 in patients with LN need to be further understood.
MicroRNAs (miRNAs) are a class of highly conserved noncoding RNAs composed of 21-25 nucleotides and have been identified as key regulators in various diseases, including SLE, by binding to target mRNA. Zeng et al. confirmed the roles of miR-371b-5p and miR-5100 in the serum of patients with SLE. 10 Fang and colleagues have reported the relationship between miR-146a and SLE. 11 Furthermore, miR-153-3p has been reported to induce immune dysregulation by inhibiting PELI1 expression in patients with SLE, 12 which indicates that miR-153-3p may be a new therapeutic target for the treatment of LN. However, the roles and molecular mechanism of miR-153-3p in LN remain unclear.
Thus, this study was designed to illustrate the role of lncRNA TUG1 in LN pathogenesis. We hypothesized that (I) lipopolysaccharide (LPS)-stimulated human renal mesangial cells (HRMCs) could be used to establish LN models in vitro; (II) lncRNA TUG1 has a protective effect on LPS-induced HRMCs; and (III) the potential mechanism of lncRNA TUG1 protection may be linked to the miR-153-3p/Bcl-2 axis. Our results may provide a novel therapy for patients with LN.

| Cell culture
HRMCs were obtained from the American Type Culture Collection (ATCC) and grown in Mesenchymal Stem Cell Medium (Thermo Fisher), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in humidified conditions with 5% CO 2 at 37°C. HRMCs (5 × 10 4 cells/well) were seeded into the 6-well plates, and when the confluence of the cells reached 80%-90%, HRMCs were then treated with 10 μg/mL LPS for 8 h for follow-up assays.

| Cell viability assay
HRMCs were seeded in 96-well plates at 37°C, treated with 10 μL 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT; 5 mg/mL) solution, and cultured for further 4 h. The solution was then removed and 100 μL DMSO was added to each well to lyse the cells in the dark. Finally, the optical density (OD) at 490 nm was measured using a multifunctional plate reader (BioTek) following the manufacturer's protocol.

| Flow cytometry analysis
After treatment, HRMC apoptosis was detected using the Annexin-V/PI Apoptosis Detection Kit (Beyotime) at room temperature for 10 min in the dark, following the manufacturer's instructions. Finally, the apoptotic cells were determined using a flow cytometer (BD Technologies) and analyzed using the Kaluza Analysis (version 2.1.1.20653; Beckman Coulter, Inc).

| Western blotting
Total proteins were extracted from HRMCs using RIPA buffer (Beyotime) and quantified using the BCA Protein Assay Kit (Invitrogen). The samples were then separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk in Phosphate buffer solution-Tween (PBST) for 1 h, the membranes were incubated with primary antibodies against β-actin, Bax, and Bcl-2 (1:1000 dilution) overnight at 4°C. Subsequently, the membranes were incubated with secondary antibodies for 1 h. Finally, the protein signals were assessed using the electrochemiluminescence (ECL) method according to the manufacturer's instructions.

| Enzyme-linked immunosorbent assay (ELISA)
After treatment, samples of the supernatant from HRMCs were collected, and the secretion levels of IL-1β, IL-6, and TNF-α were detected using ELISA kits (BD biosciences) according to the manufacturer's instructions. The OD value at 450 nm was measured on Multiskan Spectrum.

| Statistical analysis
The SPSS 20.0 software was used for statistical analysis. All results are presented as the mean ± standard deviation (SD) from three independent experiments. Statistical significance among groups was calculated using one-way analysis of variance (ANOVA) or Student's t-test. *p < .05 and **p < .01 were considered to indicate statistical significancy.

| LncRNA TUG1 directly sponges miR-153-3p
Considering that miRNAs usually act as regulators of lncRNAs to mediate gene expression, we used the starBase database to predict the target gene. We observed that lncRNA TUG1 harbored a binding site for miR-153-3p ( Figure 1A). In addition, results obtained from the dualluciferase reporter assay demonstrated that miR-153-3p-WT 3′-UTR with lncRNA TUG1 mimic remarkably suppressed the relative luciferase activity, whereas the luciferase activity was unaffected by miR-153-3p-MUT ( Figure 1B), suggesting that lncRNA TUG1 sponged miR-153-3p in the progression of LN.

| LncRNA TUG1 expression was downregulated and miR-153-3p expression was upregulated in LPS-induced HRMCs
We then analyzed the biological functions of HRMCs after treatment with 10 μg/mL LPS for 8 h. MTT and flow cytometry analysis suggested that treatment with LPS gradually inhibited cell viability ( Figure 2A) and induced apoptosis compared to the control group ( Figure 2B,C). In addition, the expression level of Bcl-2 was reduced ( Figure 2D,E), whereas Bax expression was upregulated ( Figure 2D-F) after LPS treatment of HRMCs cells compared with those in the control group. ELISA demonstrated that the secretion of inflammatory factors (TNF-α, IL-1β, and IL-6) was obviously increased in LPSinduced HRMCs ( Figure 2G-I), compared to that in the control group. Our data are consistent with a previous report that showed that LPS led to decreased lncRNA TUG1 expression and enhanced miR-153-3p levels in HRMCs ( Figure 2J,K), implying that lncRNA TUG1 and miR-153-3p are involved in LN in an in vitro model.

| MiR-153-3p negatively regulates Bcl-2 expression in HRMCs by targeting Bcl-2
To elucidate the underlying mechanisms of miR-153-3p in HRMCs, we used the online database TargetScan and identified Bcl-2 as a candidate target of miR-153-3p ( Figure 5A). A subsequent dual-luciferase reporter gene assay verified that the miR-153-3p mimic significantly reduced Bcl-2-WT luciferase activity but had no significant effect in Bcl-2-MUT group ( Figure 5B). In addition, compared to the inhibitor control group, the miR-153-3p inhibitor remarkably suppressed the level of miR-153-3p in HRMCs ( Figure 5C). RT-qPCR analysis revealed that Bcl-2 expression was suppressed after silencing Bcl-2 and upregulated in HRMCs after treatment with miR-153-3p inhibitor ( Figure 5D-F). Our data demonstrate that miR-153-3p directly sponges Bcl-2 to regulate the expression of Bcl-2 in LN.

| DISCUSSION
LN, a complex autoimmune disease caused by SLE, is one of the most fatal complications of SLE worldwide. 13 Nearly 60% of patients with SLE have LN, which can cause a series of complications, such as B-cell lymphoma, renal dysfunction, and heart and vascular problems. 14,15 To date, little is known about the nosogenesis of LN, and no satisfying therapy for LN is currently available. Therefore, understanding the pathogenesis of LN is of great significance for the development of effective therapies. Accumulating evidence has demonstrated that the dysregulation of lncRNAs is tightly associated with the occurrence of LN. Zhang et al. 16 revealed that lncRNA NEAT1 accelerates HRMC injury through the miR-146b/ TRAF6/NF-kappaB axis in LN. LncRNA TUG1 has been reported to play vital roles in multiple diseases, including atherosclerosis, 17 hepatoblastoma, 18 and colorectal cancer. 19 However, the detailed role of lncRNA TUG1 in LN remains unknown. In this study, we focused on  20 Su et al. 21 suggested that lncRNA TUG1 mediates ischemic myocardial injury by targeting the miR-132-3p/ HDAC3 axis. In this study, we first identified the target gene of lncRNA TUG1 and revealed that miR-153-3p directly interacted with lncRNA TUG1. Previous studies have indicated that dysregulated miRNA expression in cells may be involved in the pathogenesis of LN. 22 In this study, we clarified the functions of lncRNA TUG1 in LN, and the levels of lncRNA TUG1 and miR-153-3p in LPSinduced HRMCs were determined using RT-qPCR. Our data indicated that lncRNA TUG1 was downregulated and miR-153-3p was overexpressed in patients with LN and in LPS-induced HRMCs, demonstrating that lncRNA TUG1 is involved in LN progression by targeting miR-153-3p.
LPS has been used to stimulate cells to generate in vitro models. 23 In our study, HRMCs were stimulated with 10 μg/mL LPS for 8 h to conduct the LN model in vitro. Our data suggests that LPS treatment gradually inhibited HRMC viability, induced higher numbers of apoptotic cells than those in the control group, reduced Bcl-2 expression, enhanced Bax level, and promoted the secretion of inflammatory factors, in comparison with the control group, consistent with a previous study 24 ; these findings indicate that the in vitro LN model was established successfully. Our data also showed that LPS led to decreased lncRNA TUG1 expression and enhanced miR-153-3p levels in HRMCs, implying that lncRNA TUG1 and miR-153-3p are involved in LN. A large number of studies have suggested that lncRNAs are involved in various biological processes, including cell proliferation, apoptosis, and invasion. Yao et al. reported that lncRNA TUG1 knockdown repressed the viability, migration, and differentiation of osteoblasts by sponging miR-214. 25 A study by Zhang et al. 26 demonstrated that silencing lncRNA TUG1 inhibits acute myeloid leukemia cell viability and promotes apoptosis by targeting the microRNA-221-3p/KIT axis. We performed functional analysis of TUG1-plasmid or miR-153-3p mimic to explore whether lncRNA TUG1 mediated the functions of LPS in HRMCs and conducted a rescue experiment. HRMCs were transfected with controlplasmid, TUG1-plasmid, mimic control, or miR-153-3p mimic, and stimulated with 10 μg/mL LPS. We found that lncRNA TUG1 negatively regulates miR-153-3p expression in HRMCs. In addition, we found that TUG1-plasmid alleviated LPS-induced HRMC damage, as evidenced by increased cell viability, reduced numbers of apoptotic cells, enhanced Bcl-2 expression, and reduced Bax levels. However, these results were successfully reversed after transfection with miR-153-3p mimic. Inflammatory factors also exert critical roles in the development of LN. Kanno et al demonstrated that alpha2AP is related to the development of LN through the regulation of inflammatory responses. 27 We too assessed the release of the inflammatory cytokines IL-1β, IL-6, and TNF-α in LPS-induced HRMCs. ELISA showed that the TUG1-plasmid reduced the release of inflammatory factors in HRMCs, revealing that TUG1 alleviated the LPS-induced inflammatory response in HRMCs. Thus, the inhibition of apoptosis and inflammatory response in HRMCs may be beneficial for LN treatment.
Abnormal expression of miRNAs has been identified in many diseases, including LN, and miRNA function is mediated by binding to different mRNAs. 28 To further uncover the mechanism of miR-153-3p in LN, the online database TargetScan and a dual-luciferase reporter system were used to explain the relationship between miR-153-3p and Bcl-2. Further, RT-qPCR analysis revealed that the levels of Bcl-2 were high in miR-153-3p inhibitor-transfected cells, but remarkably low after Bcl-2 inhibition. Functional assays revealed a novel finding that miR-153-3p regulates HRMC proliferation, apoptosis, and inflammatory response through direct repression of Bcl-2.
Based on the above results, lncRNA TUG1 blocks LN progression by inhibiting apoptosis and inflammatory responses in LPS-stimulated HRMCs via the miR-153-3p/ Bcl-2 axis. Our findings provide new insights into LN progression and may be regarded as a promising foundation to elucidate the molecular pathogenesis of SLE.